CN113173902B - Continuous production method of oligomeric proanthocyanidins with uniform polymerization degree - Google Patents
Continuous production method of oligomeric proanthocyanidins with uniform polymerization degree Download PDFInfo
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
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Abstract
The invention relates to a continuous production method of oligomeric proanthocyanidins with uniform polymerization degree, which comprises the following steps: (1) preparing catalytic degradation liquid, catalytic degradation, membrane separation, enrichment, recycling, concentration and drying to obtain the oligomeric proanthocyanidins, wherein the obtained product has high yield, good purity and uniform polymerization degree. The method skillfully utilizes the utilization mode of the resin and the solvent circulation mode, the utilization rate of the resin and the solvent is far higher than that of the prior process for preparing the oligomeric proanthocyanidins, the process is simple and convenient, the operation is easy, no complex or large-scale equipment is needed, and the method is suitable for industrial production.
Description
Technical Field
The invention relates to a method for preparing oligomeric proanthocyanidins, and in particular relates to a continuous production method of oligomeric proanthocyanidins with uniform polymerization degree.
Background
Procyanidine is a general name of a large class of natural polyacid compounds widely existing in nature. The procyanidine has wide resource distribution, and is contained in plants such as fructus Vitis Viniferae, fructus crataegi, cacao bean, strawberry, fructus Pruni Pseudocerasi, fructus Mangifera Indicae, fructus Mali Pumilae, cypress, birch, semen Ginkgo, folium Camelliae sinensis, jowar, fructus Hordei vulgaris, Japanese Thuja, Thuja Dolabrata, herba Oriental Selaginellae, Larix Gmelini, receptaculum Nelumbinis, herba Phlomidis Regelii, and fructus Pruni Pseudocerasi.
Anthocyanin is the most effective natural antioxidant which is internationally recognized at present and is used for eliminating free radicals in human bodies, has extremely strong antioxidant and free radical eliminating capabilities which are about 20 times of Vc and 50 times of Ve, is widely used in various common foods such as cakes, cheese and the like in Europe and America, and serves as a nutrition enhancer and a natural preservative to replace a synthetic preservative. Procyanidine is used as a raw material of health food and is directly prepared into dosage forms such as capsules, and the like, and becomes one of the ten popular products of natural plant medicines in the United states. High quality procyanidins have good solubility in water and alcohol, and are widely used in beverages and wines in addition to their bright color.
Proanthocyanidins (PC) are a common polyphenolic flavone, widely found in natural plants, such as grape seeds, which are rich in Proanthocyanidins. Research shows that the grape seed procyanidin has good antioxidant function, can well remove free radicals, can treat diseases caused by the free radicals, and has the effects of resisting toxicity, diminishing inflammation, preventing and treating cardiovascular diseases and the like. Procyanidins can be classified into 3 types according to the difference in degree of polymerization: the monomer with polymerization degree of 1 includes epicatechin, gallic acid, catechin, etc., the monomer with polymerization degree of 2-4 is called oligomeric procyanidin, and the monomer with polymerization degree of not less than 5 is called polymeric procyanidin. It has been found that proanthocyanidins are absorbed by intestinal microorganisms, and the decomposition is only for monomers and dimers, and is extremely low when the molecular weight is high. The possible reason is that the polymeric procyanidin has a larger molecular weight than the oligomer and is more affected by steric hindrance, and thus the antioxidant ability of the polymer is lower than that of the oligomer. Therefore, in order to maximize the utilization efficiency of procyanidins, it is necessary to perform a degradation treatment on the polymeric procyanidins to improve the use value thereof.
There are many disclosures in the prior art for preparing oligomeric procyanidins, and the focus of product attention is mainly on average degree of polymerization, purity, and yield. For the average polymer, procyanidin monomers are generally used as a standard substance, the average polymerization degree is calculated through the mass concentration of a sample substance to be detected/the mass concentration of the sample substance to be detected, and the product components are rarely studied in detail.
The inventor's prior patent CN202110284690.8 discloses a method for continuously preparing oligomeric procyanidin, and when the inventor tests antioxidant activity of oligomeric procyanidin products with approximate average polymerization degree, the inventor finds that even though the average polymerization degree is the same, the antioxidant activity is greatly different. The reason for this may be that the average polymerization degrees of different products are close to each other due to the discrete polymerization degrees, but the average polymerization degree is calculated from the data of the total amount of the substances, and thus the uniformity of the procyanidin oligomer product cannot be characterized, and it can also be understood that the molecular weight distribution of the procyanidin oligomer product cannot be characterized. Therefore, even if the average polymerization degree is the same, the ratio of the products having a polymerization degree of 2 to 3 may be different, resulting in different antioxidant activity. It is well known that the uniformity of the ingredients, quality and performance of medicinal products is a key factor for evaluating the reliability of medicaments,
CN101100464A discloses an oligomeric proanthocyanidin with high ORAC value and a purification method thereof, and specifically, an oligomeric anthocyanin product with different ORAC values is obtained by dissolving a raw material containing proanthocyanidin, performing ultrafiltration, and selecting an ultrafiltration membrane without copper retention component. However, the method is actually a screening method for different molecular weights of products, and the quality of the products is not uniform by adjusting the preparation process.
CN103923052A discloses a preparation method of oligomeric proanthocyanidins, which comprises the steps of extracting by 60-70% ethanol, controlling the pH value to be 4.0-4.4, and eluting by utilizing the principles of perforated resin adsorption and different solubilities of proanthocyanidins with different polymerization degrees in ethanol with different concentrations, wherein the obtained product has high content of oligomeric products with the polymerization degree of 2-4. The product obtained by the method has low average polymerization degree, and the content of oligomeric procyanidin in the product is more than 95%. However, this method rejects large amounts of other ethanol concentrations and does not improve the homogeneity of the product by the production process, so that the yield is only 56% to 78%.
CN109943605A discloses a method for preparing oligomeric proanthocyanidins with uniform components, which comprises fermenting raw material aqueous solution containing proanthocyanidins, extracting, degreasing, extracting, drying to obtain crude product, passing through resin column, eluting, and lyophilizing to obtain oligomeric proanthocyanidins product. The polymerization degree is uniform, and the average polymerization degree is small. The patented method is characterized in that 5 wt% methanol, 30 wt% methanol, 70 wt% methanol and 95 wt% methanol are respectively eluted, 70% methanol aqueous solution is collected to obtain a product which is oligomeric proanthocyanidins with uniform components, the essence of the method is that the product obtained by degrading high polymeric proanthocyanidins is selectively adsorbed, and the yield is not high.
Moreover, none of the above methods relates to a continuous production method of procyanidin oligomer. Therefore, it is of great significance to develop a method capable of continuously producing the oligomeric proanthocyanidins while having uniform product components.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art and providing the method for continuously preparing the oligomeric proanthocyanidins, and meanwhile, the obtained product has high polymerization degree uniformity, stable product quality, simple process, low requirement on equipment and strong industrialization capability, and can realize continuous operation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a continuous production method of oligomeric proanthocyanidins with uniform polymerization degree comprises the following steps:
(1) preparing a catalytic degradation liquid: dissolving a raw material containing high polymeric proanthocyanidins in an alkaline catalytic degradation liquid, and stirring until the raw material is fully dissolved;
(2) and (3) catalytic degradation: loading the degradation liquid on an anion exchange resin, and collecting effluent liquid I;
(3) membrane separation: passing the effluent I through a membrane, and respectively collecting the trapped fluid and the permeate;
(4) enrichment: loading the permeate into a macroporous adsorption resin column until the permeate is saturated, replacing the macroporous adsorption resin with the same specification and the same type, continuously loading the permeate into the column, removing impurities from the saturated macroporous adsorption resin after removing water, and adding an alcohol water solution for desorption for later use; when the permeate is loaded on another macroporous adsorption resin column to saturation, replacing the macroporous adsorption resin for water removal, impurity removal and desorption for standby, continuously loading the macroporous adsorption resin column to saturation, circularly and alternately using more than two resins, respectively collecting and combining effluent liquid II loaded on the column and desorption liquid, and adding organic weak acid during desorption;
(5) and (3) recycling: mixing the effluent liquid II in the step (4) with the trapped liquid in the step (3), adding a raw material containing high polymeric proanthocyanidins for dissolving, and then supplementing and applying the dissolved raw material as the degradation liquid in the step (1), and continuously loading the mixture into a column according to the method in the step (2);
(6) concentrating and drying: and (4) concentrating and drying the desorption solution obtained in the step (4) to obtain the oligomeric proanthocyanidins product.
In the step (1), the raw material containing high polymeric proanthocyanidins contains proanthocyanidins mixture with a polymeric degree of more than or equal to 5 and a high polymeric anthocyanin content of more than 60 wt%. The procyanidin-containing material is prepared by extracting and refining plant materials, including but not limited to cranberry procyanidin, cowberry procyanidin, roselle procyanidin, grape seed procyanidin, sphenoidea frutescens procyanidin, blueberry procyanidin, purple sweet potato procyanidin, purple rice procyanidin or black rice procyanidin.
In the step (1), the catalytic degradation liquid is a mixture of alkali, ethyl acetate, ethanol and water; preferably, the mass ratio of ethyl acetate, ethanol and water is 0.1-0.5: 0.5-2: 10-15, wherein the addition amount of the alkali is that the pH value of the catalytic degradation liquid is 10-14, and preferably 12-13; the base comprises a metal hydroxide, preferably a metal carbonate and/or a metal bicarbonate.
Further, the metal hydroxide is at least one of sodium hydroxide, potassium hydroxide and barium hydroxide; the metal carbonate is at least one of sodium carbonate and potassium carbonate, and the metal bicarbonate is at least one of sodium bicarbonate and potassium bicarbonate.
Further, the metal hydroxide is a mixed alkali of sodium hydroxide/potassium hydroxide and barium hydroxide; preferably mixed alkali of sodium hydroxide/potassium hydroxide and barium hydroxide according to the mass ratio of 2-10: 1; more preferably, the metal hydroxide is sodium hydroxide/potassium hydroxide and barium hydroxide according to a mass ratio of 4-7: 1. The inventors have unexpectedly found that the degradation efficiency of the polymeric procyanidin can be further enhanced and more oligomers can be generated by using the mixed alkali of sodium hydroxide/potassium hydroxide and barium hydroxide. Presumably, the reason is that, in addition to the barium hydroxide being able to act as a base, the metal barium ions may also have some effect on the degradation.
Furthermore, the alkali also comprises metal carbonate and/or metal bicarbonate, and the mass ratio of the metal carbonate and/or the metal bicarbonate to the metal hydroxide is 1: 1-3. Applicants have found that a basic degradation system contains a certain amount of weak acid anions that contribute to the stability of the degradation.
In the step (1), the ratio of the raw material containing high polymeric proanthocyanidin to the catalytic degradation liquid is such that the concentration of the raw material containing high polymeric proanthocyanidin in the solution is 30-100g/L, preferably 50-70 g/L.
In the step (2), the anion exchange resin is selected from LX-T5, LXD-762, LX-94 and D941, the volume of the anion exchange resin is 0.1 to 0.3 time, preferably 0.2 to 0.25 time of that of the catalytic degradation liquid, the diameter-height ratio of the chromatographic column is 1:3-8, and the flow rate of the upper column is 0.1 to 0.5 BV/h. The anion exchange resin provides a relatively stable alkaline condition similar to that of the buffer solution, and is favorable for stable and uniform degradation of the proanthocyanidin polymer.
In the step (2), the temperature of the anion exchange resin column is kept between 40 and 50 ℃. In this temperature range, the proanthocyanidin high polymer is stably and uniformly degraded.
In the step (3), the membrane is preferably a rotating cross-flow filtration membrane, and compared with the conventional static membrane filtration, the flux of the rotating cross-flow filtration membrane is large, so that the column is not easy to block, and the interception molecular weight of the rotating cross-flow filtration membrane is 1000-3000 Da.
In the step (4), the number of the two or more macroporous adsorption resins is 2-10, and the conditions such as the amount of raw materials and the treatment capacity of the resin column can be flexibly adjusted according to actual conditions, for example, the method is suitable for alternately using 2, 3, 4 and 5 macroporous adsorption resins.
The macroporous adsorbent resin is any one of D101, AB-8, DM130, LSA-10, LX-12, XDA-6, XDA-7, LX-68 and XDA-200B; the volume of the macroporous adsorption resin is 0.1 to 0.3 times of that of the catalytic degradation liquid; the diameter-height ratio of the chromatographic column is 1:3-8, and the flow rate of the chromatographic column is 0.5-1 BV/h.
In the step (4), 0.7-1.5BV of pure water is used for water driving, and the flow rate is 1-2 BV/h; the impurity removal is to use 2-3BV of low-degree ethyl acetate/alcohol aqueous solution with the flow rate of 0.5-1BV/h, wherein the volume fraction of ethyl acetate is 2-4% and the volume fraction of alcohol is 5-10% in the low-degree ethyl acetate/alcohol aqueous solution, and the alcohol is at least one of ethanol and methanol.
In the step (4), the desorption is carried out by eluting with 2-3BV of 40-70% volume fraction alcohol-water solution, the alcohol-water solution also contains organic weak acid, and the addition amount of organic acid is such that the pH value of the alcohol-water desorption is 3-6, preferably 4-5; the flow rate is 0.5-1 BV/h; the alcohol is at least one of ethanol and methanol, and the weak organic acid is selected from at least one of malic acid, citric acid and ascorbic acid. The acidic condition is favorable for maintaining the stability of the procyanidin.
In the step (4), the effluent liquid II from the column loading process is the effluent liquid from the column loading process, and does not include the effluent liquid from the water driving process and the effluent liquid from impurity removal process.
In the step (5), the adding amount of the raw material containing high polymeric proanthocyanidins enables the mass concentration of the raw material containing high polymeric proanthocyanidins in the combined degradation liquid to be close to the concentration in the catalytic degradation liquid prepared in the step (1), namely 30-100g/L, preferably 50-70g/L, and the pH value of the combined degradation liquid is adjusted to be close to the pH value of the catalytic degradation liquid prepared in the step (1) by alkali. The alkali is consistent with the catalytic degradation liquid prepared in the step (1).
In the step (6), the concentration is to reduce water, and comprises but is not limited to one of vacuum rotary concentration, single-effect reduced pressure evaporation concentration and multi-effect reduced pressure evaporation concentration, wherein the concentration temperature is not higher than 70 ℃, and the vacuum degree is-0.06 to-0.1 MPa; the drying is to remove water, and includes but not includes any one of vacuum drying, forced air drying, vacuum microwave drying and vacuum freeze drying, and the drying temperature is not higher than 70 ℃.
The principle of the method of the invention is as follows:
dissolving a certain amount of high polymeric proanthocyanidins in a catalytic degradation liquid (a mixture of ethyl acetate, alcohol, water and alkali), loading the proanthocyanidins degraded to different degrees onto an anion exchange resin, wherein the weak base anions of the anion exchange resin provide a more stable alkaline degradation environment, which can be helpful for obtaining oligomeric proanthocyanidins products with uniform relative polymerization degree, and then separating effluent liquid containing the oligomeric proanthocyanidins through a rupture membrane again to realize separation of the proanthocyanidins with different polymerization degrees; and then loading the membrane permeate rich in oligomeric proanthocyanidins to macroporous adsorption resin, performing water washing and desorption after adsorption saturation, and adding a certain amount of organic weak acid during desorption to improve the stability of oligomers during desorption. The invention replaces more than 2 macroporous absorption resins of the same type, and the macroporous absorption resins are alternately used after being saturated. Concentrating and drying the collected desorption solution to obtain the oligomeric proanthocyanidins. The trapped fluid obtained by membrane separation is combined with the effluent liquid of the anion exchange resin on the column for reuse, namely, the high polymeric proanthocyanidins are added again to be used as a new catalytic degradation liquid for continuous degradation and are loaded on the anion exchange column, thereby realizing the continuity of the whole production process. The obtained product has high purity, good overall yield and uniform polymerization degree, and is a continuous production method of the oligomeric proanthocyanidins product with uniform polymerization degree.
The method has the following beneficial effects:
the method is a method for continuously producing the oligomeric proanthocyanidins product and is convenient for industrial production, skillfully utilizes a resin utilization mode and a solvent circulation mode, the utilization rate of the resin and the solvent is far higher than that of the conventional process for preparing the oligomeric proanthocyanidins, the process is simple, convenient and easy to operate, complex or large-scale equipment is not required, and the method is suitable for industrial production. The obtained oligomeric proanthocyanidin product has high quality and high yield, and has industrial advantages.
The invention provides a relatively stable alkaline degradation environment by the alkaline catalytic degradation condition, particularly the degradation under the anion exchange resin, and the degradation liquid contains a certain amount of weak alkaline anions, so that the procyanidin high polymer is stably and continuously degraded into a product with relatively uniform polymerization degree.
Drawings
FIG. 1 is a schematic flow diagram of the preparation of oligomeric procyanidins of the invention.
Detailed Description
The present invention will be further described with reference to the following examples.
The high-polymer procyanidin product used in the embodiment of the invention is provided by Jiangxi Haichi rich bioengineering GmbH, the average polymerization degree of grape seed procyanidin is 11.6, and the content is 76.4 wt%.
The starting materials or chemical reagents used or the equipment used in the examples of the present invention are commercially available in a conventional manner unless otherwise specified.
The rotary cross-flow filter membrane is purchased from environment-friendly technology Limited company FLD-SYS-1000 of Fulinde Nanjing, and has a molecular weight cutoff of 1000 Da.
Anion exchange resins were purchased from new materials science and technology, Inc., of Xian blue, dawn.
The detection method used in the present invention is detailed as follows:
1. the detection method of the average polymerization degree comprises the following steps:
1) determination of mass concentration of substances: preparing catechin methanol reference substances with the concentrations of 30, 90, 150, 210 and 270 mu g/ml respectively, accurately weighing 0.5ml of the reference substances, placing the reference substances in 10ml glass test tubes with plugs respectively, then accurately adding 3ml of 4% vanillin methanol solution and 1.5ml of concentrated hydrochloric acid respectively, shaking up, carrying out water bath at 20 ℃ for 15min, measuring the absorbance at the wavelength of 500nm and making a standard curve. And (3) accurately weighing a certain amount of sample to be detected to replace the standard substance by the same method, configuring and detecting by the method, and calculating through a standard curve to obtain the mass concentration of the sample to be detected.
2) Determination of the quantitative concentration of the substance: accurately measuring appropriate amount of catechin reference substance solution, placing into 5ml measuring flask, adding acetic acid solution (containing 2% methanol) to scale, respectively making into 0.025, 0.050, 0.075, 0.100, 0.125 μmol/ml reference substance solution, placing 1ml into 10ml test tube with plug, adding 4% hydrochloric acid and 1.0% vanillin acetic acid solution 5ml, shaking, water bathing at 20 deg.C for 20min, measuring absorbance at 500nm wavelength and making standard curve. Diluting the sample diluent to be detected in the step 1) into a certain concentration multiple by the same method, detecting by the above method instead of a reference substance, and calculating by a standard curve and the dilution multiple to obtain the quantitative concentration of the sample substance to be detected.
3) Measurement of average polymerization degree: the average polymerization degree (mass concentration of the sample substance to be measured/mass concentration of the sample substance to be measured)/molar mass of the control.
2. Content determination method
After the mass concentration of the sample to be detected is obtained in the method (1), the anthocyanin content can be obtained according to the dilution ratio of the sample.
3. Method for measuring molecular weight and molecular weight distribution
Gel permeation chromatography (Shanghai Huishi GPC102 molecular weight detection gel)Permeameter), oligomeric procyanidins with different polymerization degrees and approximate molecular weights are selected as standard substances, because procyanidin oligomers with different polymerization degrees and known molecular weights are not purchased in the market. Drawing a linear regression equation of a standard curve: lgMw＝a+btR，MwIs the weight average molecular weight of the standard sample, tRIs the retention time. The molecular weight of haploid catechin is 290.26, the molecular weight of dimeric procyanidin B2 is 578.53, the molecular weight of trimeric procyanidin C1 is 886.77, and the molecular weight of procyanidin tetramer I is 1153.68.
Injecting a sample to be detected into a chromatograph, and calculating according to the following formula to obtain the molecular weight and the molecular weight distribution:
D＝Mw/Mn
RIi is the peak height of the sample at retention time i, and Mi is the molecular weight of the sample at retention time i.
Example 1
(1) Preparing a catalytic degradation liquid: 4000g of grape seed procyanidin is taken, 500g of grape seed procyanidin is firstly taken, and the volume ratio of the grape seed procyanidin to the 10L of ethyl acetate, ethanol and water is 0.4: 1: 10, adjusting the pH value to 13 by using alkali, wherein the alkali is sodium hydroxide, and the mass ratio of barium hydroxide to sodium carbonate is 8: 2: 5 in a mixture.
(2) And (3) catalytic degradation: loading the catalytic degradation liquid on a 100-mesh 2L anion exchange resin column LX-T5 with a diameter-height ratio of 1:4, loading the bottom end of the column with a flow rate of 0.1BV/h and a column temperature of 50 ℃, collecting effluent liquid I, and keeping the temperature of the anion exchange resin column at 45 +/-1 ℃ through an interlayer outside the column during degradation.
(3) Membrane filtration: and (3) passing the effluent liquid I through a rotating cross-flow filtering membrane with the molecular weight cutoff of 1000Da, and respectively collecting the cutoff liquid and the permeate liquid.
(4) Enrichment: and (3) loading the permeate into a 2L D101 macroporous adsorption resin column, wherein the height ratio of the chromatographic column diameter is 1:4, the flow rate is 0.5BV/h, and replacing the D101 macroporous adsorption resin with the same specification when the permeate is saturated. After the adsorption saturated chromatographic column is washed by 1BV of pure water at the flow rate of 1BV/h, 2.5BV of low-alcohol ethyl acetate alcohol water solvent is used for removing impurities (the volume fraction of ethyl acetate is 2 percent, the volume fraction of ethanol is 6 percent), and the flow rate is 1 BV/h; and finally, desorbing by using an ethanol water solution with the volume fraction of 3BV and the percentage of 60%, wherein the ethanol water solution contains malic acid, the pH value is adjusted to be 5.5, the flow rate is 1BV/h, the desorbed macroporous adsorption resin can be continuously loaded onto a column until the macroporous adsorption resin is saturated, and the two loaded columns of the D101 macroporous adsorption resin, namely the effluent II and the desorption liquid, are respectively combined.
(5) Mechanically applying: and (3) mixing the effluent liquid II in the step (4) and the trapped liquid in the step (3), adding high polymeric proanthocyanidin to dissolve until the concentration is 5%, and adding sodium hydroxide, barium hydroxide and sodium carbonate according to a mass ratio of 8: 2: and (5) after the pH value is adjusted to 13 by the mixed alkali, continuously returning to the step (2) for loading.
(6) Concentrating and drying: and (5) concentrating the desorption solution obtained in the step (4) to 45brix under the conditions of 65 ℃ and-0.09 MPa in vacuum, and then drying the desorption solution under the conditions of 70 ℃ and-0.09 MPa in vacuum to obtain 2963g of oligomeric procyanidin product.
The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidins product is 3.6, the molecular weight distribution D is 1.82, the content is 94.52 percent, and the yield is 91.64 percent.
Example 2
The other operations and conditions were the same as in example 1 except that in the step (1), the pH of the catalytic degradation solution was adjusted to 12 with a base. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidins product is 3.7, the molecular weight distribution D is 1.79, the content is 95.09 percent, and the yield is 90.83 percent.
Example 3
The other operations and conditions were the same as in example 1 except that in step (1), the pH of the catalytic degradation solution was adjusted to 14 with a base. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidins product is 3.4, the molecular weight distribution D is 1.94, the content is 91.25 percent, and the yield is 90.83 percent.
Example 4
The other operations and conditions are the same as those in example 1, except that in step (1), the base for adjusting the pH of the catalytic degradation liquid is potassium hydroxide, and the ratio of barium hydroxide to potassium bicarbonate is 7: 1:4 in the presence of a catalyst. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidin product is 3.6, the molecular weight distribution D is 1.84, the content is 94.27 percent, and the yield is 91.71 percent.
Example 5
The other operations and conditions were the same as in example 1 except that in step (2), the anion exchange resin LX-T5 was replaced with an anion exchange resin LXD-762. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidin product is 3.5, the molecular weight distribution D is 1.85, the content is 94.31%, and the yield is 91.48%.
Example 6
The other operations and conditions were the same as in example 1 except that in step (4), the alcohol aqueous solution contained 6 wt% ascorbic acid and no other weak organic acid at the time of desorption. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidin product is 3.5, the molecular weight distribution D is 1.87, the content is 94.45 percent, and the yield is 91.52 percent.
Example 7
The other operations and conditions were the same as in example 1 except that in step (1), the base for adjusting the pH of the catalytic degradation liquid was sodium hydroxide, and barium hydroxide was mixed with the metal hydroxide in a mass ratio of 4: 1. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidin product is 3.5, the molecular weight distribution D is 1.93, the content is 94.61%, and the yield is 91.73%.
Example 8
The other operations and conditions were the same as in example 1 except that in step (1), the base for adjusting the pH of the catalytic degradation liquid was sodium hydroxide. The detection calculation results are that: the average polymerization degree of the oligomeric proanthocyanidins product is 3.5, the molecular weight distribution D is 1.92, the content is 93.25 percent, and the yield is 88.71 percent.
Comparative example 1
The other operations and conditions were the same as in example 1 except that the anion exchange resin in step (2) was replaced with a polyamide resin having a particle size of about 150. mu.m. The detection calculation results in that: in this example, the average degree of polymerization of the oligomeric procyanidin product was 3.5, the molecular weight distribution D was 2.14, the content was 94.22%, and the yield was 93.53%.
Comparative example 2
The other operations and conditions were the same as in example 1 except that in step (4), the alcohol aqueous solution contained no malic acid upon desorption. The detection calculation results are that: in this example, the average degree of polymerization of the oligomeric procyanidin product was 3.5, the molecular weight distribution D was 2.08, the content was 92.73%, and the yield was 91.28%.
Application exampleMeasurement of antioxidant Activity
The method adopts a DPPH method to test the in-vitro antioxidant activity of a sample, and specifically comprises the steps of preparing the samples of the examples and the comparative examples into ethanol solutions with different mass concentrations for later use, preparing the DPPH into a 0.3mM solution by using sewage ethanol, adding 5mL of the sample solution into a 5mL of the DPPPH solution, shaking up, keeping out of the sun, placing in a water bath at 40 ℃ for 30min, and testing the absorbance at 517 nm. The concentration IC of the sample with the DPPH clearance rate of 50 percent is calculated according to the DPPH standard curve50(mg/L). And testing the IC of vitamin C (Vc) under the same conditions50As a positive control. The results are shown in table 1 below:
as can be seen from the antioxidant activity of the examples and comparative examples of the invention, the oligomeric proanthocyanidin product prepared by the invention has stronger in-vitro antioxidant activity and more uniform polymerization degree under the same or similar average polymerization degree. The possible reason is that the content of effective oligomer (polymerization degree of 2-3) in the oligomeric proanthocyanidin product obtained by the method is higher. The oligomeric proanthocyanidin product can be obtained by a continuous process, has uniform polymerization degree and stable quality, and is a production process of the oligomeric proanthocyanidin with great industrial prospect.
Claims (12)
1. A continuous production method of oligomeric proanthocyanidins with uniform polymerization degree comprises the following steps:
(1) preparing a catalytic degradation liquid: dissolving a raw material containing high polymeric proanthocyanidins in an alkaline catalytic degradation liquid, and stirring until the raw material is fully dissolved; the catalytic degradation liquid is a mixture of alkali, ethyl acetate, ethanol and water, and the addition amount of the alkali is such that the pH value of the catalytic degradation liquid is 12-13; the alkali comprises metal hydroxide, metal carbonate and/or metal bicarbonate, and the metal hydroxide is mixed alkali of sodium hydroxide/potassium hydroxide and barium hydroxide according to the mass ratio of 2-10: 1;
(2) and (3) catalytic degradation: loading the degradation liquid on an anion exchange resin containing weak base anions, and collecting effluent liquid I;
(3) membrane separation: passing the effluent I through a membrane, and respectively collecting the trapped fluid and the permeate;
(4) enrichment: loading the permeate into a macroporous adsorption resin column until the permeate is saturated, replacing the macroporous adsorption resin with the same specification and the same type, continuously loading the permeate into the column, removing impurities from the saturated macroporous adsorption resin after removing water, and adding an alcohol water solution for desorption for later use; when the permeate is loaded on another macroporous adsorption resin column to saturation, replacing the macroporous adsorption resin for water removal, impurity removal and desorption for standby, continuously loading the macroporous adsorption resin column to saturation, circularly and alternately using more than two resins, respectively collecting and combining effluent liquid II loaded on the column and desorption liquid, and adding organic weak acid during desorption; the organic weak acid is selected from at least one of malic acid, citric acid and ascorbic acid;
(5) and (3) recycling: mixing the effluent liquid II in the step (4) with the trapped liquid in the step (3), adding a raw material containing high polymeric proanthocyanidins for dissolving, and then supplementing and applying the dissolved raw material as the degradation liquid in the step (1), and continuously loading the mixture into a column according to the method in the step (2);
(6) concentrating and drying: and (4) concentrating and drying the desorption solution obtained in the step (4) to obtain the oligomeric proanthocyanidins product.
2. The continuous production method according to claim 1, wherein the catalytic degradation liquid in the step (1) has a mass ratio of ethyl acetate, ethanol and water of 0.1-0.5: 0.5-2: 10-15.
3. The continuous production process according to claim 1, wherein the metal carbonate is at least one of sodium carbonate and potassium carbonate, and the metal bicarbonate is at least one of sodium bicarbonate and potassium bicarbonate.
4. The continuous production method according to claim 1, wherein the metal hydroxide is sodium hydroxide/potassium hydroxide, and the ratio of barium hydroxide is 4-7: 1.
5. The continuous production method according to claim 1, wherein the mass ratio of the metal carbonate and/or the metal bicarbonate to the metal hydroxide is 1: 1-3.
6. The continuous production method as claimed in claim 1, wherein in the step (1), the ratio of the high polymeric proanthocyanidin-containing material to the catalytic degradation liquid is such that the concentration of the high polymeric proanthocyanidin-containing material in the solution is 30-100 g/L.
7. The continuous production method as claimed in claim 6, wherein the ratio of the high polymeric proanthocyanidin-containing material to the catalytic degradation liquid is such that the concentration of the high polymeric proanthocyanidin-containing material in the solution is 50-70 g/L.
8. The continuous production method according to claim 1, wherein in the step (2), the anion exchange resin is selected from LX-T5, LXD-762, LX-94 and D941, the volume of the anion exchange resin is 0.1 to 0.3 times that of the catalytic degradation liquid, the diameter-height ratio of the anion exchange resin column is 1:3-8, and the flow rate of the upper column is 0.1-0.5 BV/h; the temperature of the anion exchange resin column is kept between 40 and 50 ℃.
9. The continuous production method according to claim 8, wherein the volume of the anion exchange resin is 0.2 to 0.25 times that of the catalytic degradation liquid.
10. The continuous production method according to claim 1, wherein in the step (4), the number of two or more macroporous adsorbent resins is 2 to 10; the volume of the macroporous adsorption resin is 0.1 to 0.3 times of that of the catalytic degradation liquid; the diameter-height ratio of the macroporous adsorption resin column is 1:3-8, and the flow rate of the macroporous adsorption resin column is 0.5-1 BV/h; the water driving is performed by using 0.7-1.5BV of pure water with the flow rate of 1-2 BV/h; the impurity removal is to use 2-3BV of low-degree ethyl acetate/alcohol aqueous solution with the flow rate of 0.5-1BV/h, wherein in the low-degree ethyl acetate/alcohol aqueous solution, the volume fraction of ethyl acetate is 2-4%, the volume fraction of alcohol is 5-10%, and the alcohol is at least one of ethanol and methanol; the desorption is carried out by eluting with 2-3BV of 40-70% alcohol-water solution by volume fraction.
11. The continuous production method according to claim 10, wherein the organic acid is added in an amount such that the pH at which the alcohol and water are desorbed is 3 to 6; the flow rate is 0.5-1 BV/h; the alcohol is at least one of ethanol and methanol.
12. The continuous production method according to claim 11, wherein the organic acid is added in an amount such that the pH at which the alcohol and water are desorbed is 4 to 5.
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